Use of nf-kappa b inhibitors for the treatment of mastitis

ABSTRACT

The present invention relates to the use of NF-KB (NF-kappa B) inhibitors for the manufacture of a medicament for the treatment of mastitis.

The present invention relates to the use of NF-κB (NF-kappa B)inhibitors for the manufacture of a medicament for the treatment ofmastitis.

Mastitis is one of the most costly diseases in dairy herds. Costs mainlyarise from decreased milk production and quality, therapeuticinterventions, loss of antibiotic-contaminated milk and extra labor.Mastitis, defined as an inflammatory condition of the mammary gland, isusually caused by pathogenic bacteria. The most common pathogens thatprovoke mastitis include Staphylococcus aureus, Streptococcusagalactiae, Streptococcus dysgalactiae, Streptococcus uberis,Arcanobacterium pyogenes and coliforms. These microorganisms can invadethe udder through the teat canal and produce inflammation of themilk-producing tissue causing the formation of scar tissue which, onceformed, may cause a permanent reduction in the cow's milk production. Aninfection can also alter the composition, quantity, appearance andquality of the milk.

Mastitis in dairy cattle is usually treated with antibiotics. However ithas been found to be desirable to replace such treatment withantibiotics with treatment by non-antibiotic chemo-therapeutic drugs toavoid building up of resistance of bacterial strains. Furthermore, milkproduced by dairy cattle on an antibiotic treatment has to be discardedas it contains residual antibiotics.

NF-κB (nuclear factor-kappa B) is a collective name for inducibledimeric transcription factors composed of members of the Rel family ofDNA-binding proteins that recognize a common sequence motif. NF-κB isfound in essentially all cell types and is involved in activation of anexceptionally large number of genes in response to infections,inflammation, and other stressful situations requiring rapidreprogramming of gene expression.

Surprisingly, it has been found that compounds capable of inhibiting theactivation of NF-κB, i.e. NF-κB inhibitors, can be of use in thetreatment of mastitis.

Known NF-κB inhibitors are e.g.:

-   -   15d-PGJ2 (15-deoxy-Δ.12,14-prostaglandin J2) (see Proc. Natl.        Acad. Sci. USA, 97(9), 4844-4849 (2000));    -   tepoxalin (see Immunology Letters, 53(2,3), 109-113 (1996));    -   cycloepoxydon and its (+)- and (−)-stereoisomers (see J. Am.        Chem. Soc., 123(45), 11308-11309 (2001));    -   sodium diethyldithiocarbamate (Na-DDTC) (see Brain Research        Bulletin, 55/3, 375-386 (2001));    -   gliotoxin, MG-132        (N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-Leucinamide),        BAY 11-7082        (3-[[4-(1,1-dimethylethyl)phenyl]-sulfonyl]-2-propenenitrile),        BAY 11-7085        ((E)-(3-[[4-(1,1-dimethylethyl)phenyl]-sulfonyl]-2-propenenitrile),        (see Human Pathology, 31(12), 1482-1490 (2000));    -   cyclopentenone prostaglandin A1 (PGA1), (see Proc. Natl. Acad.        Sci. USA, 94, 746-750 (1997));    -   cyclopentabenzofurans as described in WO-00/08007; and    -   [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]-amino]butyl]-boronic        acid also known under the INN name of bortezomib.

Other NF-κB inhibitors can be learned from literature and compounds canbe tested for NF-κB inhibiting activity using assays known in the art.See for instance Proc. Natl. Acad. Sci. USA, 94, 746-750 (1997).

The NF-κB inhibitors for use in the treatment of mastitis can bespecific inhibitors of NF-κB or can be non-specific.

The present invention provides the use of a NF-κB inhibitor for themanufacture of a medicament for the treatment of mastitis.

Consequently, the present invention provides a method for treatingmastitis comprising administering to a subject an effective amount of aNF-κB inhibitor.

As used herein, the term “subject” embraces warm-blooded animal speciessuch as e.g. ruminants such as cattle, sheep, goats, buffalo etc.

There are a variety of forms or types of bovine mastitis, with varyingseverity and symptomatology, including the following:

-   -   acute mastitis which is an abrupt and short-lived inflammation        of the udder,    -   chronic mastitis which is a moderate and persistent inflammation        of the mammary gland.

Acute mastitis is generally a clinical form whereas chronic mastitis isoften a subclinical form of the disease. Subclinical mastitis is a formof mastitis in which there is no swelling of the gland or observableabnormality of the milk, although there are changes in the milk, such asan increase in somatic cell count (SCC), that can be detected by specialtests. This type of mastitis is by far the most prevalent and causes thegreatest overall loss in most herds. It often is referred to as “hidden”mastitis. Clinical mastitis is a form of mastitis in which the abnormalconditions of the udder and secretion are observable. Mild clinicalmastitis involves changes in the milk such as flakes, clots, and awatery or unusual appearance. Heat and sensitiveness of the udder areslight or absent, but there may be signs of swelling. Severe clinicalmastitis involves a sudden onset with swelling of the infected quarterwhich is hot, hard and sensitive. The milk appears abnormal and milkproduction drops. Sometimes, in addition to the local effects in theudder, the cow herself becomes sick. There are signs of fever, rapidpulse, depression, weakness and loss of appetite. The combination ofthese conditions often is referred to as acute systemic mastitis,because not only the udder, but the whole animal is affected.

The effective amount of NF-κB inhibitor for the treatment of mastitiswill be determined using routine optimization techniques and aredependent upon the particular condition to be treated, the condition ofthe subject, the route of administration, the formulation, and thejudgment of the practitioner and other factors evident to those skilledin the art. An effective amount may be achieved by multiple dosing.

Compositions comprising a NF-κB inhibitor may be administeredintravascularly, intraperitoneally, subcutaneously, intramuscularly, orby intramammary infusion using forms known in the pharmaceutical art.For intravascular, intraperitoneal, subcutaneous, intramuscular orintramammary administration, active drug components may be combined witha suitable carrier such as water, saline, aqueous dextrose, and thelike. Veterinary formulations comprising a NF-κB inhibitor are usuallyprepared by mixing said NF-κB inhibitor with an excipient, diluting byan excipient or enclosing within a carrier that can be in the form of acapsule, sachet, or other container. When the excipient serves as adiluent, it can be a solid, semi-solid, or liquid material that acts asa vehicle, carrier or medium for the NF-κB inhibitor. Examples ofsuitable excipients include lactose, dextrose, sucrose, sorbitol,mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxybenzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated to provide quick,sustained or delayed release of the active ingredient afteradministration to the host by procedures known in the art.

Intramammary infusions may be oil based, e.g. a vegetable oil such aspeanut oil or a non-vegetable oil such as mineral oil and may furtherinclude a thickening agent and optionally also a surfactant.

Compositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, infusions,syrups, aerosols, ointments, soft and hard gelatin capsules,suppositories, sterile injectable solutions and sterile packagespowders. Regardless of the route of administration selected, thesecompositions are formulated into pharmaceutically acceptable dosageforms by conventional methods known to those skilled in the art.

The NF-κB inhibitors may also be used in combination therapy withantibiotics. Antibiotics used in the treatment of mastitis are e.g.beta-lactam antibiotics such as ampicillin, cloxacillin, hetacillin,nafcillin, penicillin G, procaine penicillin; cephalosporins such ascefoperazone, cefuroxime, cefalonium, cefapirin, cefoxazole,cefracetrile; aminoglycoside antibiotics such as framycetin, neomycin,novobiocin, streptomycin; macrolide antibiotics such as erythromycin;tetracyclines such as chlortetracycline, oxytetracycline; or polypeptideantibiotics such as polymyxin B.

Experimental Details

The level of NF-κB activity in milk cells from healthy and acute andchronic mastitis-affected cows was measured.

A. Measurement of NF-κB Activity in Mastitis Affected Cows

1. Animals

Twenty healthy, six acute and twenty chronic mastitis-affectedHolstein-Friesian cows were used in this study. Healthy cows had lowsomatic cell count (SCC) (<10⁵ cells/ml) and were free of any clinicalsign of mastitis. Acute mastitis-affected cows had high SCC (>4×10⁵cells/ml) and exhibited clinical signs of illness. Neither healthy noracute mastitis-affected cows had earlier record of udder disease, asmeasured by clinical symptoms and monthly SCC recordings. Chronicmastitis-affected cows had persistently increased SCC (>4×10⁵ cells/ml),as determined by monthly SCC measurements, but were devoid of anyclinical sign of the disease. All the cows were between 2 and 7 yearsold, had a lactation number ranging from 1 to 5 and were lactating for 1to 6 months. None of the cows received any treatment during the monthpreceding the experiments.

2. Sterile Milk Sampling

Milk was collected using a sterile teat cannula infusion apparatus. Thisapparatus consisted of a sterile and pyrogen-free cannula inserted intothe teat canal and connected to the free end of an infusion set attachedto a 2000 ml sterile collection bag. Milk samples were taken from onequarter. Before sampling, the quarter was thoroughly disinfected with asolution containing 70% alcohol and 30% chlorohexidine. Immediatelyafter milk sampling, an aliquot of 50 μl was the subject of amicrobiological analysis. Samples that were found to be contaminated bybacteria were excluded from the study.

3. Somatic Cell Count

Immediately after milk collection, 50 ml of each milk sample was shippedto a specialized laboratory (Laboratory of the Milk Committee, Battice,Belgium), where a somatic cell count was performed following classicalprocedure.

4. Isolation of Milk Cells

Milk samples were first centrifuged (300 g) for 30 minutes at 4° C.Pellets were then washed in phosphate buffered saline (PBS) andcentrifuged (300 g) for another 30 minutes at 4° C. The pelleted cellswere finally resuspended in 1 ml of PBS before protein extraction. Celldifferentials were performed on cytospin preparations stained withDiff-Quick (Dade Behring, Dudingen, Germany).

5. Protein Extraction

Isolated milk cells were centrifuged for 10 minutes at 300 g and thepellet was resuspended in 600 μl lysis buffer (0.1% nonidet P-40; 10 mMTris HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl₂; 1 mM EDTA; 2 mM orthovanadate;1 mM diisopropyl fluorophosphates (DFP); 10 μg/ml leupeptin; 10 μg/mlaprotinin). The cells were vortexed 15 seconds, kept on ice for 5minutes, and centrifuged for 10 minutes at 300 g. The resulting pelletwas resuspended in a KCl buffer (10 mM Hepes, pH 7.4; 400 mM KCl; 10%Glycerol; 2 mM EDTA; 1 mM Ethylene glycol-bis[β-aminoethyl ether]-N,N,N′N′-tetraacetic acid (EGTA); 1% nonidet P-40, 1 mM dithiothreitol(DTT); 2 mM orthovanadate; 10 μg/ml leupeptin; 10 μg/ml aprotinin; 1 mMDFP) and kept at 4° C. for 10 minutes before centrifugation for 15minutes at 12,000 g. The supernatant was diluted three times and storedat −80° C. until use.

6. NF-κB Electrophoretic Mobility Shift Assays (EMSAs)

Binding reactions were performed for 30 minutes at room temperature with5 μg of total protein extracts in 20 mM Hepes (pH 7.9), 10 mM KCl, 0.2mM EDTA, 20% (v/v) glycerol, 1% (w/v) acetylated bovine serum albumin, 3μg of poly(dI-dC) (Amersham Pharmacia Biotech, Aylesbury, U.K.), 1 mMDTT (dithiothreitol), 1 mM phenylmethylsulfonyl fluoride, and 100,000cpm of ³²P-labeled double-stranded oligonucleotide probes. Probes wereprepared by annealing the appropriate single-stranded oligonucleotides(Eurogentech, Liège, Belgium) at 65° C. for 10 minutes in 10 mM Tris, 1mM EDTA, and 10 mM NaCl, followed by slow cooling to room temperature.The probes were labeled by end-filling with the Klenow fragment of E.coli DNA polymerase I (Roche, Mannheim, Germany), with [³²P]dATP and[³²P]dCTP (Dupont-New England Nuclear, Les Ulis, Fance). Labeled probeswere purified by spin chromatography on Sephadex G-25 columns (Roche).DNA-protein complexes were separated from unbound probe on 4% nativepolyacrylamide gels at 150 V in 0.25 M Tris, 0.25 M sodium borate, and0.5 mM EDTA, pH 8.0. Gels were vacuum-dried and exposed to x-ray film at−80° C. for 12 hours. The amount of specific complexes was determined byphotodensitometry of the autoradiography. To confirm specificity,competition assays were performed with a 50-fold excess of unlabeledwild-type and mutated probes. For supershift experiments, 1.5 μl of eachantibody was incubated with the extracts 30 minutes before addition ofthe radiolabeled probe. The sequences of the oligonucleotides used inthis work were as follows: wild-type palindromic κB probe, 5′-TTG GCAACG GCA GGG GAA TTC CCC TCT CCT TAG GTT-3′; mutated palindromic κBprobe, 5′-TTG GCA ACG GCA GAT CTA TTC CCC TCT CCT TAG GTT-3′. The NF-κBactivity was assessed by photodensitometry of the specific bands.

7. Results

Active NF-κB complexes were undetectable in milk cells from healthycows, as determined by EMSAs. Conversely, protein extracts prepared frommilk samples of acute mastitis-affected cows demonstrated a high NF-κBactivity. In milk cells from cows suffering from chronic mastitis, NF-κBactivity varied from low to high.

Linear regressions were carried out to assess the relation between thelevel of NF-κB activity in milk cells, as assessed by photodensitometry,and the degree of inflammation in the mammary gland. This regressionanalysis demonstrated a highly significant (P<0.01) correlation betweenNF-κB activity and two markers of udder inflammation, namely SCC (FIG.1A) and the percentage of neutrophils in milk samples (FIG. 1B).

This study demonstrated that NF-κB activity was increased in milk cellsfrom mastitis-affected cows and therefore NF-κB inhibitors are believedto be of potential use in the treatment of mastitis.

B. Effects of Administration of an NF-κB Inhibitor on Milk CellsObtained From Chronic Mastitis-Affected Cows

1. Animals

Four chronic mastitis-affected Holstein-Friesian cows were used in thisstudy. These cows had persistently increased SCC (>4×10⁵ cells/ml), asdetermined by monthly SCC measurements, but were devoid of any clinicalsign of the disease. None of the cows received any treatment during themonth preceding the experiments.

2. Sterile Milk Sampling

Milk was collected as described above.

3. Somatic Cell Count

Immediately after milk collection, 50 ml of each milk sample was shippedto a specialized laboratory (Laboratory of the Milk Committee, Battice,Belgium), where a somatic cell count was performed following classicalprocedure.

4. Isolation of Milk Cells

Milk samples were first centrifuged (300 g) for 30 minutes at 4° C.Pellets were then washed in phosphate buffered saline (PBS) andcentrifuged (300 g) for another 30 minutes at 4° C. The pelleted cellswere finally resuspended in 1 ml of PBS before protein extraction. Celldifferentials were performed on cytospin preparations stained withDiff-Quick (Dade Behring, Dudingen, Germany).

5. Culture and Treatment of Milk Cells

Milk cells were cultured at 4×10⁶ per ml in RPMI 1640 medium (LifeTechnologies, Merelbeke, Belgium) supplemented with 1% glutamine, 10%foetal bovine serum, streptomycine 50 μg/ml, and penicillin 50 UI/ml.The cells were first incubated at 37° C. in a 5% CO2-95% air mixture for90 minutes then treated with various doses of NF-κB inhibitors (15-dPGJ2at concentrations of 40 μM, 50 μM, 60 μM and 80 μM; or gliotoxine at aconcentration of 1 μl/ml). Treated cells were incubated 3 hours beforeprotein extraction.

6. Protein Extraction

See procedure described above.

7. NF-κB Electrophoretic Mobility Shift Assays (EMSAs)

See procedure described above for assessing NF-κB activity byphotodensitometry.

8. Results

In untreated milk cells from chronic mastitis-affected cows, an elevatedlevel of NF-κB activity was detected, as determined by EMSAs.Conversely, protein extracts prepared from milk cells treated with15d-PGJ2 or glyotoxine demonstrated a reduced NF-κB activity.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relation between specific NF-κB activity displayed bymilk cells, as determined by photodensitometry, and SCC (A) and thepercentage of neutrophils (B) in healthy (▪, n=6) and acute (●, n=6) andchronic (▴, n=20) mastitis-affected Holstein-Friesian cows. r,correlation coefficient.

FIG. 2: 15d-PGJ2 and gliotoxine inhibit NF-κB activation in milk cellsfrom chronic mastitis-affected cows. After isolation, milk cells wereincubated for 90 min and then treated with the indicated concentrationsof 15d-PGJ2 and gliotoxine. Protein extracts were assessed 3 h after thetreatment for NF-κB DNA-binding activity by EMSAs.

1. (canceled)
 2. The method of claim 8 wherein mastitis is chronicmastitis.
 3. The method of claim 8 wherein mastitis is acute mastitis.4. (canceled)
 5. The method of claim 8 wherein the NF-κB inhibitor is aspecific NF-κB inhibitor.
 6. The method of claim 8 wherein the NF-κBinhibitor is selected from 15d-PGJ2, tepoxalin, cycloepoxydon,(+)-cycloepoxydon, (−)-cyclo-epoxydon, sodium diethyldithiocarbamate,gliotoxin, MG-132, BAY 11-7082, BAY 11-7085 or bortezomib.
 7. The methodof claim 8 wherein the NF-κB inhibitor is administered intramammary. 8.A method for the treatment of mastitis comprising administering to asubject in need thereof an effective amount of a NF-κB inhibitor.
 9. Amethod for the treatment of mastitis comprising administering to asubject in need thereof an effective amount of a NF-κB inhibitor incombination with an antibiotic.
 10. The method of claim 2, wherein theNF-κB inhibitor is selected from 15d-PGJ2, tepoxalin, cycloepoxydon,(+)-cycloepoxydon, (−)-cyclo-epoxydon, sodium diethyldithiocarbamate,gliotoxin, MG-132, BAY 11-7082, BAY 11-7085 or bortezomib.
 11. Themethod of claim 3, wherein the NF-κB inhibitor is selected from15d-PGJ2, tepoxalin, cycloepoxydon, (+)-cycloepoxydon,(−)-cyclo-epoxydon, sodium diethyldithiocarbamate, gliotoxin, MG-132,BAY 11-7082, BAY 11-7085 or bortezomib.